The alphaviruses are a group of 25 related viruses, many of which are important human or veterinary pathogens, including several viruses endemic to the United States that give rise to periodic epidemics of encephalitis. We propose to continue our studies of the molecular biology of virus replication and on molecular aspects of interaction of the virus with the immune system and of neurovirulence, using primarily a genetic approach with the type alphavirus, Sindbis virus. The functions of various domains of the structural proteins will be probed by mapping temperature-sensitive mutants of Sindbis virus that are defective in the capsid protein autoprotease, in glycoprotein synthesis, processing, and transport, or in virus assembly. The domains of glycoprotein E2 forming their major neutralization epitope and possibly important for neurovirulence will be probed by mapping variants selected to be resistant to monoclonal antibodies or to be neurovirulent for mice. Preliminary findings suggest that the neutralization epitopes are located in a relatively short region of E2 and all involve charged amino acids. The specific interaction of this region of E2 with various cell types appears to determine alphavirus tissue tropisms as well. Small differences in the distribution of charged residues on the surface of the virion apparently can result in very different patterns of pathogenesis in whole animals. These studies will be augmented with studies of variants constructed by the methods of site-specific mutagenesis and testing the activity of the altered proteins in in vivo or in vitro expression systems. Studies of RNA sites involved in virus assembly will be performed with ts103, a mutant defective in virus assembly. We will also study the functions of the nonstructural proteins required for viral RNA replication and transcription. These will include mapping of ts lesions so that the four nonstructural complementation groups can be assigned to specific proteins, use of monospecific antisera to study the synthesis, processing and interactions amoung the nonstructural proteins, and use of site-specific mutagenesis to alter selected regions of these proteins. We will also continue our comparative studies of alphaviruses using Middelburg virus, looking for regions of particular homology in the nonstructural proteins, in an attempt to understand evolution within the alphavirus genus and the relationship of alphaviruses to other groups of RNA viruses.